In scanning microscopy, a specimen is illuminated with a light beam in order to observe the detected light (in the form of reflected or fluorescent light) emitted by the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detected light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the current mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detection of the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels via the beam deflection device back to the beam splitter, passes through the latter and is then focused onto the detection pinhole, behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detected light that does not originate directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which, by sequential scanning of the specimen with the focus of the illuminating light beam, results in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers. Commercial scanning microscopes usually comprise a scan module that is flange-mounted onto the stand of a conventional light microscope and contains all the aforesaid elements additionally necessary for scanning a specimen.
Commercial scanning microscopes usually contain a microscope stand like the one also used in conventional light microscopy. As a rule, confocal scanning microscopes in particular are also usable as conventional light microscopes. In conventional fluorescent incident-light microscopy, the portion of the light of a light source (for example of an arc lamp) that comprises the desired wavelength region for fluorescent excitation is coupled into the microscope beam path with the aid of a color filter called the excitation filter. Coupling into the beam path of the microscope is accomplished using a dichroic beam splitter, which reflects the excitation light to the specimen while allowing the fluorescent light proceeding from the specimen to pass largely unimpeded. The excitation light scattered back from the specimen is held back with a blocking filter that is, however, transparent to the fluorescent radiation. Combining mutually matched filters and beam splitters optimally to yield an easily interchangeable modular filter block has been common for some time. The filter blocks are usually arranged in a turret within the microscope as a part of so-called fluorescent incident-light illuminators, thus enabling rapid and easy interchanging.
In confocal scanning microscopy, a detection pinhole can be dispensed in the case of two-photon (or multi-photon) excitation, since the excitation probability depends on the square of the photon density and thus on the square of the illuminating light intensity, which of course is much greater at the focus than in the adjacent regions. The fluorescent light being detected therefore very probably originates almost exclusively from the focus region, which renders superfluous any further differentiation, using a pinhole arrangement, between fluorescent photons from the focus region and fluorescent photons from the adjacent regions.
Especially given that the yield of fluorescent photons in two-photon excitation is in any case low, a non-descan arrangement, in which the detected light does not arrive at the detector via the beam deflection device (descan arrangement) and the beam splitter for coupling in the illuminating light, but rather is deflected directly after the objective by means of a dichroic beam splitter and detected, is of interest because less light is generally lost when the detected light is guided in this fashion. In addition, when descan detection is used in two-photon excitation, scattered components of the detected light contribute significantly to the signal, whereas with non-descan detection they play only a greatly reduced role. Arrangements of this kind are known, for example, from the publication by David W. Piston et al., “Two-photon excitation fluorescence imaging of three-dimensional calcium ion activity,” Applied Optics, Vol. 33, No. 4, February 1996, and from Piston et al., “Time-Resolved Fluorescence Imaging and Background Rejection by Two-Photon Excitation in Laser Scanning Microscopy,” SPIE Vol. 1640.
One problem with the known arrangements is that of arranging a beam splitter, to deflect the detected light out of the microscope beam path after the objective, within a scanning microscope for non-descan deflection, and aligning it precisely. This requires the implementation of complex additional arrangements that necessitate massive physical modifications to the scanning microscope and in particular to the microscope stand. Retrofitting to a scanning microscope with descan detection is usually impossible or very complex.